We wish to develop an understanding of signaling networks in the retinal pigment epithelium (RPE) with special emphasis on retinoid metabolism pathways and protection against light damage and oxidative stress. Apoptotic RPE cell death resulting from increased oxidative stress could hasten the onset of age-related macular degeneration (AMD) and may be regulated by retinoic acid (RA). RA affects many cellular functions including cell growth, differentiation, and apoptosis. Synthetic analogs of retinoic acid also have significant effects on cellular function. One such analog, fenretinide (N-(4-hydoxyphenyl)retinamide; 4HPR), has been used as a cancer preventive agent and has been proposed as a therapeutic agent for lipofuscin-based retinal diseases, and we are interested in how these effects of 4HPR are mediated. We previously found that neuronal differentiation of ARPE-19 cells induced by 4HPR was mediated by the MAPK pathway and that this was associated with decreased expression of insulin-like growth factor binding protein-5 under the regulation of CCAAT/enhancer-binding protein. Stearoyl-CoA desaturase (SCD), a rate-limiting enzyme in the synthesis of unsaturated fatty acids, is also involved in 4HPR-induced effects on RPE cells. SCD plays an important early role in the synthesis of sphingolipids and ceramides, important effectors in cellular biology. A close association between the production of ceramide and the onset of programmed cell death has been well established. We are interested in the role of SCD in RPE biochemistry. In another avenue of regulation, microRNAs (miRNAs) have received much attention as post-transcriptional regulators of gene expression in all cell/tissue types. Given the likely importance of this level of regulation in the response of RPE cells to various signals we are interested in determining changes in miRNA expression in RPE cells due to agents with which they are treated in our experiments. We previously found that 4HPR increased expression of microRNA-9, and that inflammatory cytokines regulate microRNA-155 expression in human retinal pigment epithelial cells by activating the JAK/STAT pathway. In the past year we have made progress in the following areas: 1) We continued to study the role of SCD in 4HPR-induced apoptosis of RPE cells. Endoplasmic reticulum (ER) is the central organelle in eukaryotic cell for lipid synthesis, protein folding and maturation. A number of biochemical and physiological stimuli induces ER stress, and if unchecked could lead to apoptosis. Recombinant SCD protein is degraded via the ubiquitin-proteasome-dependent pathway when overexpressed in cells. It is also known that ER stress induces ubiquitin-proteasome-dependent degradation of proteins. We examined whether ER stress affects the SCD expression. Human ARPE-19 cells treated with 4HPR, tunicamycin or thapsigargin, compounds that induce ER stress, showed a time-dependent decrease in the expression of SCD protein, while markers of ER stress were markedly increased. The decrease in SCD protein expression was completely blocked by the proteasome inhibitor, MG132. In addition, PYR-41, an irreversible inhibitor of ubiquitin activating enzyme E1, also completely blocked the 4HPR-induced decrease in SCD protein expression. Immunoprecipitation analysis of 4HPR-treated cell lysate using either ubiquitin or SCD antibody showed that ER stress increased the ubiquitination of proteins including SCD. These data show that ER stress mediates the degradation of SCD in human RPE cells via the ubiquitin-proteasome dependent pathway. A manuscript describing these data is currently in preparation. 2) We have continued our work on the role of miRNAs in regulating the inflammatory response of adult human RPE cells. Inflammatory response of the RPE is implicated in the pathogenesis of age-related macular degeneration (AMD). The microRNAs miR-146a and miR-146b-5p are known to control the inflammatory process by their ability to regulate key genes involved in cytokine signaling by translational repression. We have investigated the expression of miR-146a and miR-146b-5p in human RPE cells and their response to pro-inflammatory cytokines. Real-time PCR analysis of confluent cultures of RPE cells established from adult human donor eyes showed that miR-146a and 146b-5p are expressed in RPE cells. The cells responded to pro-inflammatory cytokines (IFN-&#947; + TNF-&#945; + IL-1&#946;) by highly increasing the expression of both miR-146a and miR-146b-5p. This was associated with an increase in the expression of transcripts for CCL2, CCL5, CXCL9, CXCL10 and IL-6, and a decrease in that for HMOX1. The miR-146a induction was dependent on IL-1&#946; since its omission from the cytokine mix resulted in a greatly reduced response. In contrast, the induction of miR-146b-5p was dependent on IFN-&#947; since its omission from the cytokine mix minimized the effect. Also, the increase in MIR146B promoter activity by the cytokine mix was effectively blocked by JAK inhibitor 1, a known inhibitor of JAK/STAT signaling pathway. The expression of IRAK1 protein was decreased when ARPE-19 cells were transiently transfected with miR-146a or miR-146b-5p mimics. These results show that both miR-146a and miR-146b-5p are expressed in human RPE cells in culture and their expression is regulated by pro-inflammatory cytokines, miR-146a being dependent on IL-1&#946; and miR-146b-5p on IFN-&#947;. These two microRNAs could play a role in inflammatory processes underlying AMD or other retinal degenerative diseases by their ability to regulate IRAK1 expression. A manuscript describing these data is currently in preparation. 3) We continued a study to understand the mechanisms underlying dedifferentiation of RPE cells in primary culture. Divergence from or convergence to the phenotype of native RPE is a common theme of much RPE cell culture research. On the one hand, induced pluripotent stem (iPS) cells can be differentiated into cells sharing many aspects of RPE phenotype, and by rigorous culture methods, fetal RPE cells can be differentiated to retain or acquire aspects of native phenotype. On the other hand, explanted native RPE cells will lose important aspects of their RPE phenotype after a short time in culture. The various immortalized cell lines, such as the commonly used ARPE-19, have lost most native phenotypic features. What are the mechanisms regulating such gain or loss? Do mechanisms like epithelial-mesenchyme transition play a role in this process? We are particularly interested in the long-known but poorly understood loss by immortalized and primary RPE cells of expression of visual cycle enzymes. Understanding the mechanism underlying this down-regulation could be useful in ensuring that iPS-derived cells used for human transplant are fully competent to fulfill their intended role in restoring RPE function in treated eyes. Our experimental paradigm focuses on the loss of visual cycle competence by adult bovine RPE cells explanted into primary culture. We have developed methods for establishing bovine RPE cells in primary culture. Using these we are analyzing expression of visual cycle and other genes and will correlate these to changes in gene regulation, RNA transcript expression and microRNA expression patterns. The study is still ongoing. 4) We have continued analysis of post-transcriptional modifications of IRBP. We have established non-radioactive HPLC/mass spectrometric assays for several fatty acids, sphingolipids and ceramides to assist in current research. In addition, we have collaborated within the LRCMB and with other laboratories and sections (LI, Molecular Structure and Functional Genomics) to provide retinoid and other analyses.